Phosphorus is an essential element for the growth of all organisms. In livestock production, phosphorus deficiency has been described as the most prevalent mineral deficiency throughout the world and feed must often be supplemented with inorganic phosphorus in order to obtain desired growth performance of monogastric animals (e.g. pigs, poultry etc.).
Phytic acid, or phytate, (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) is a major storage form of phosphorus in cereals and legumes, representing 18% to 88% of the total phosphorus content (Reddy et al. 1982). The enzyme phytase (myo-inositol hexakisphosphate phosphohydrolase) belongs to the group of phosphoric monoester hydrolases: it catalyzes the hydrolysis of phytate (myo-inositol hexakis phosphate) to inorganic monophosphate and lower phosphoric esters of myo-inositol or, in some cases, free myo-inositol. Phytases are classified either as 3-phytases or 6-phytases based on the first phosphate group attacked by the enzyme. 3-phytase is typical for microorganisms and 6-phytase for plants (Cosgrove, 1980).
Phytase is either absent or present at a very low levels in monogastric animals (Bitar and Reinhold 1972; Iqbal et al. 1994). Consequently, dietary phytate is not digested or absorbed from the small intestine and instead is concentrated in fecal material, thereby contributing to phosphorus pollution in areas of intensive livestock production. Runoff from animal farms leads to contamination of rivers and streams. Such runoff has resulted in rapid drops in the oxygen concentration in rivers and streams due to excessive algal growth in water, which, in turn, has led to an increase in the mortality rate of fish and existing flora and fauna. This is becoming a global problem as pig and poultry production is increased (Miner 1999; Mallin 2000). Furthermore, phytic acid is viewed as an anti-nutritional factor because it interacts with essential dietary minerals and proteins limiting the nutritional values of cereals and legumes in man and animals (Harland and Morris 1995).
For the above reasons, various attempts have been made to enable animals to utilize available phytate in feed. Such attempts have included production of low phytate plants (Abelson 1999), addition of phytase to the animal feed (Simons et al. 1990) (Stahl et al. 1999) or transformation of the fodder plants to produce the required phytase (Pen et al. 1993, Verwoerd et al. 1995). A combination of these options, the feeding of phytase to poultry receiving low phytate corn has also been tested (Huff et al. 1998). However, these solutions increase the cost of animal production. Also because phytase is an enzyme, it is susceptible to inactivation by heat and moisture and is generally unstable at the high temperatures used for feed pelleting.
The primary phytase used for supplementing animal feeds is from Asperigillus sp.; however, phytases are produced by a large number of plants and microorganisms (Wodzinski and Ullah 1996) (Dvorakova 1998). A phytase produced by Escherichia coli has been reported to exhibit the highest activity of those reported (Wodzinski and Ullah 1996). This phytase from E. coli was initially cloned as an acid phosphatase gene that was designated APPA (Dassa et al. 1990). Greiner et al. (1991; 1993) purified phytase from E. coli and reported that some of the kinetic properties of the acid phosphatase activity of the native phytase of E. coli were similar to those of the APPA-encoded acid phosphatase. However, the authors did not clone the phytase gene to prove that it was identical to APPA gene. We have subsequently cloned, overexpressed and characterized APPA gene, and shown that the E. coli gene APPA codes for a bifunctional enzyme exhibiting both phytase and acid phosphatase activities (Golovan et al. 2000). Phytases exhibit phosphatase activity, however the relative activities differ widely among enzymes (Wodzinski and Ullah 1996).
Therefore, there is a need for an improved method of allowing access by animals to phytase so as to enable efficient phytate metabolism and, thereby reducing phosphate pollution.
In the field of protein production using recombinant methods, one of the associated problems relates to the lack of required glycosylation. Therefore, a method of producing such glycoproteins is also needed.